Ischemic heart disease remains as the single largest cause of mortality in the United States. Delivery of c-kit positive human cardiac stem cells (hCSCs) is a very promising therapeutic approach in repairing the infarcted heart, but is severely limited by the poor survival of donor cells. Carbon monoxide (CO), a byproduct of heme oxygenase 1 (HO-1), has a potent anti- apoptotic, cytoprotective effect against ischemia/reperfusion injury to cardiomyocytes. However, it is not known whether HO-1/CO is cytoprotective for hCSCs. Our pilot studies found that HO-1/CO plays an important role in mediating hCSC survival ability. Therefore, this application focuses on testing our hypothesis that HO-1/CO promotes human cardiac stem cell survival vial the activation of survival signal pathways and the cytokine effects, as well as enhances the stem cell therapy efficacy and heart function after transplantation into immunodeficient mouse heart following myocardial infarction. Thus, we will examine whether hCSCs pretreated with an HO-1 inducer (CoPP), or a CO releasing molecule (CORM-3), will exhibit greater abilities in cell survival, proliferation, migration, endothelial and cardiomyogenic differentiation (Aim 1), and understand the molecular mechanisms that underlie the effect of HO-1/CO on hCSC survival signal pathways, resistance to apoptosis, ROS generation and cytokine release (Aim 2). Aim 3 of this proposal will test whether transplantation of the preconditioned eGFP+-hCSCs will result in improvement in invivo hCSC survival and homing, endogenous mouse CSC proliferation and differentiation, and cardiac structure and function with immunodeficient mouse (SCID) model following myocardial infarction. Knowledge gained from the above experiments will provide the essential implications on the strategies to enhance hCSC survival after transplantation and, therefore, their efficacy in clinical repairing infarcted myocardium for the cell therapy of patients with ischemic heart disease. PUBLIC HEALTH RELEVANCE: Poor survival of donor cells is one of the major challenges in stem cell therapy for ischemic heart disease. This project investigates the effective strategies to enhance the human cardiac stem cell therapy efficacy with small molecules, including cobalt protoporphyrin (CoPP), a heme oxygenase 1 inducer, which promotes endogenous carbon monoxide generation; or a carbon monoxide donor, CORM-3. These studies will show how CoPP/CORM-3 affects the survival ability of human cardiac stem cells in an in vitro cell culture model, and how intramyocardial injection of CoPP/CORM-3 pretreated human cardiac stem cells improve heart function in an in vivo immunodeficient mouse model following ischemia-reperfusion injury. The results and broad conclusions will be directly relevant to enhance the efficacy in clinical repairing the infarcted myocardium for the cell therapy of patients with ischemic heart disease.